One of the directions of development in quantitative phase imaging is to provide the capability to reconstruct the phase or preferably refractive index (RI) distribution within thick, highly scattering samples. This direction coincides with current trends in biology, where three-dimensional (3D) organoids are currently replacing standard 2D cultures as more physiological models for tissue growth and organ formation in a dish. The biological complexity of these 3D structures makes the imaging and RI reconstruction particularly challenging, and thus calibration as well as validation structures are important and sought-after tools in instrumentation development. For this reason, in this work, we present the full preparation and measurement procedure for organoid phantoms printed with two-photon polymerization along with the method to obtain the ground truth of the object structure independently of RI reconstruction errors and artifacts.
In this work we present a phase-retrieval-based approach to quantifying the diffusion of drugs through biomembranes or biofilms. So far, the phenomenon was studied based on fringe orientation resulting from a refractive index gradient in the vicinity of the interface. The result is usually obtained with a Mach-Zehnder interferometer with an imaging system. This approach limits the spatial resolution of the method and does not allow observation of local changes in the diffusion. For this reason, we propose to use a single-shot phase retrieval method by utilizing a polarization-sensitive CMOS sensor to obtain four phase-shifted interferograms in a polarization-modified version of the interferometer. Finally, we demonstrate the operation of the system by quantitative analysis of ampicillin diffusion through Pseudomonas aeruginosa biofilm formed on the polyethylene terepthtalate membrane.
KEYWORDS: Microfluidics, Refractive index, Tomography, Statistical analysis, Holography, Biological research, Lab on a chip, 3D metrology, Imaging systems
Holographic tomography (HT) is a label-free, high-resolution and non-invasive method that retrieves 3D refractive index (RI) information about analysed biological specimens. The most common measurement scenario includes culturing and analysing cells directly in a Petri dish. However, it does not mimic the in vivo conditions unlike the microfluidic approach. Thus, in our work, we have focused on the development of a measurement configuration that is dedicated to analysis of cell dynamics in a lab-on-chip. It includes a fast HT system, a new ultra-thin microfluidic chip that allows for long term monitoring in controlled environment, a stitching algorithm that allows to combine single fields of view (FoV) into a synthetic field of view in three dimensions and the full volume RI analysis of internal cellular organelles during measurements. This setup provides the ability to track changes occurring in individual cell organelles as well as getting statistically valuable data. In experimental verification, A549-type and MeWo cells were cultured under microfluidic conditions in the chip and put under observation using HT.
Holographic tomography (HT) is a measurement technique utilizing refractive index (RI) as imaging contrast and enabling wide spectrum of applications in modern cell biology. Acquired 3D RI distribution, however, is strongly influenced by the measurement setup and data processing, which calls for reliable tools and methods to characterize and compare metrological parameters of the resulting reconstruction. In this paper we demonstrate and analyze the differences in reconstructions of a 3D-printed test object, which has the optical and structural features of a typical biological (mammalian) cell and has been fabricated at the sufficient level of accuracy for both the geometrical shape and RI distribution metrology. Experimental results have been acquired using commercial and research HT systems and further compared with reference data in an attempt to show the contribution of hardware and software components to the total error. The metrological performance is quantified and discussed in the context of the parameters that are usually of interest during the biomedical interpretation stage such as 3D resolution, volume, RI and dry mass of subcellular structures.
Limited angle holographic tomography (LAHT) is currently the most common tool in biomedical applications of 3D quantitative phase imaging. It uses the refractive index (RI) as contrast agent for a single cell or tissue analysis and provides highly accurate RI values in the full measurement volume. Recently several new systems have been built in laboratories and new devices have been released into the market. All of them apply algorithms and processing paths which significantly influence correctness of the results. In our work we perform study of the selected LAHT systems and compare their 3D metrological features and other functional parameters.
In holographic tomography (HT), the 3D refractive index distribution within weakly-scattering, phase-only biological object is retrieved. This key property of the technique is one of its most significant strengths compared to labelling-based methods of cell analysis such as fluorescence microscopy. As a consequence, however, it is required to acquire a set of holograms at several viewing directions, which hinders the measurement speed. In this paper we explore the prospect of multiplexing projections in order to decrease the number of scanning positions required for the full measurements. The presented analysis is based on experimental data acquired in a limited-angle holographic tomography system and emulates the performance of a spatial-light-modulator-based system in which multiple projections may be acquired simultaneously by generating a distribution of multiple point sources in the Fourier plane of the condenser lens. For this reason, the increase in acquisition speed strictly depends on the number of multiplexed holograms and results in decreased reconstruction quality. The performance of the system is demonstrated and analyzed with biological objects - human keratinocyte cells.
Holographic tomography (HT) is a measurement technique utilizing refractive index (RI) as imaging contrast and enabling wide spectrum of applications in modern cell biology. Obtained 3D RI distribution within a sample is quantitative, however it is prone to phase measurement and numerical errors especially in the case of limited angle holographic tomography (LATH). Therefore, determination and control over metrological parameters of HT system is crucial for credibility and usefulness of obtained results. In this work we propose a new type of calibration 3D phantom for LATH that allows to determine accuracy of a tomographic system. We have experimentally verified that it is possible to design, fabricate (using two-photon polymerization method) and measure complex microstructure containing regions of constant, step-like and gradient RI distribution. The phantom printing parameters required to obtain the reference RI values are determined based on its measurements using well-established 2D techniques (digital holographic microscope and white-light interferometry). The final calibration structure printed with multiple RI levels is measured by full angle HT as the reference method for LAHT. The advantages and limitations connected with implementation of the proposed phantom are discussed.
In holographic tomography obtaining projections is the key part of the measurement process that enables the 3D refractive index reconstruction. The error of the illumination angle can significantly influence the reconstruction quality and alter the result degrading the reliability of the reconstruction. Thus, in this paper the impact of the scanning errors in limited angle holographic tomography with respect to two reconstruction algorithms is analyzed. The simulated errors are compared to the errors identified in the experimental system. The reconstruction errors are verified using a paramecium cell phantom at the simulation stage and with a biological object, namely a macrophage cell in the experimental part. The experimental system presented in the paper exhibits maximum expected measurement errors found in galvanometer-mirror-based holographic tomography setups.
Limited-angle optical diffraction tomography (LAODT) is a powerful tool for measuring 3D refractive index distribution in biological microsamples. However, when thick objects are measured, reconstructions are erroneous due to diffraction errors even in the case when tomographic reconstruction algorithms take into account this phenomenon. We propose a hardware-based solution which allows to change a focal plane position with a liquid tunable lens in LAODT system. For each illumination angle, projections with different focal plane positions are recorded, and thus diffraction errors in the neighborhood of these planes are minimized. In this paper, we describe a method for processing data from a varifocal tomography setup that utilizes a Generalized Total Variation Iterative Constraint algorithm.
In this paper a new, hardware-based solution for extending the depth of field in holographic tomography is presented. The solution is based on a 4f system and an electric, focus-tunable lens, which provides fast, motion-free defocusing of the plane conjugate with the camera, which acquires holograms. The optimum parameters for the required axial scanning are provided for a specific model of a commercially available tunable lens. Then, the quality of the system equipped with the designed module is analyzed and the reconstruction of a standard object (microsphere) scanned by the 4f-based defocusing system is presented. Finally, the result of the increased depth of field in the measurement domain is demonstrated with a reconstruction of a mouse fibroblast cell.
Optical diffraction tomography has been steadily proving its potential to study one of the hot topics in modern cell biology — 3D dynamic changes in cells' morphology represented with refractive index values. In this technique digital holography is combined with tomographic reconstruction and thus it is necessary to provide projections acquired at different viewing directions. Usually the Mach-Zehnder interferometer configuration is used and while the object beam performs scanning, the reference beam is in most cases stationary. This approach either limits possible scanning strategies or requires additional mechanical movement to be introduced in the reference beam. On the other hand, spiral or grid scanning is possible in alternative common-path or Michelson configurations. However, in this case there is no guarantee that a specimen is sparse enough for the object to interfere with an object-free part of the beam. In this paper we present a modified version of Mach-Zehnder interferometer-based tomographic microscope, in which both object and reference beam are subject to scanning using one scanning device only thus making any scanning scenario possible. This concept is realized with a custom-built optical system in the reference beam and is appropriate for mechanical as well as optical scanning. Usually, the tomographic reconstruction setups and algorithms are verified using a microsphere phantom, which is not enough to test the influence of the distribution of the projections. In this work we propose a more complex calibration object created using two-photon polymerization.
We demonstrate an active, holographic tomography system, working with limited angle of projections, realized by optical-only, diffraction-based beam steering. The system created for this purpose is a Mach–Zehnder interferometer modified to serve as a digital holographic microscope with a high numerical aperture illumination module and a spatial light modulator (SLM). Such a solution is fast and robust. Apart from providing an elegant solution to viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different illumination scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied here to reconstruct nonpiecewise constant samples. Finally, the accuracy of full measurement and the proposed processing path is tested for a calibrated three-dimensional micro-object as well as a biological object—C2C12 myoblast cell.
The case of diffraction tomography with limited angle of projections is discussed from the algorithmic and experimental points of view. To reconstruct a three-dimensional distribution of refractive index of a micro-object under study, we use a hybrid approach based on the simultaneous algebraic reconstruction technique (SART) enhanced by a compressed sensing reconstruction technique. It enables us to apply the standard computed tomography algorithms (which assume that the rays are traveling in straight lines through the object) for phase data obtained by means of digital holography. We present the results of analysis of a phantom and real objects obtained by applying SART with anisotropic total variation (ATV) minimization. The real data are acquired from an experimental setup based on a Mach–Zehnder interferometer configuration. Also, it is proven that in the case of simulated data, the limited number of projections captured in a limited angular range can be compensated by a higher number of iterations of the algorithm. We also show that the SART + ATV method applied for experimental data gives better results than the data replenishment algorithm.
In the paper we demonstrate a holographic tomography system with limited angle of projections, realized by optical– only, diffraction-based beam steering. The system created for this purpose is a Mach-Zehnder interferometer modified to serve as a digital holographic microscope with high Numerical Aperture illumination module and a Spatial Light Modulator. Such solution is fast and robust. Apart from providing an elegant solution to the viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied to non-piecewise constant samples. Finally, the accuracy of full measurement and processing path proposed is tested for a calibrated 3D microobject.
In the paper the case of diffraction tomography with limited angle of projections is discussed from the experimental and algorithmic point of views. To reconstruct a 3D distribution of refractive index of an object under study, we use the hybrid approach, which enables to apply the standard Computer Tomography algorithms for phase data obtained by digital holography. We present the results of applying Simultaneous Algebraic Reconstruction Technique together with Anisotropic Total Variation minimization (SART+ATV) on both a phantom object and real data acquired from an experimental setup based on a Mach-Zehnder interferometer configuration. Also, the analysis of the influence of the limited number of projections within a limited angular range is presented. We prove that in the case of simulated data, the limited number of projections captured in a limited angular range can be compensated by higher number of iterations of the algorithm. We also show that SART+ATV method applied for experimental data gives better results than the popular Data Replenishment algorithm.
A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a “3-D cluster” as well as subcellular organelles like the nucleoli and local internal refractive index changes.
In this paper we present a method for numerical correction of phase images captured in a digital holographic microscopy (DHM) setup adapted to tomographic measurement of biological objects. The purpose of the correction is a removal of the object wave deformation associated with a fluid filled fiber capillary, which is used in DHM system to enable manipulation of a specimen. The proposed correction procedure is based on a simple concept of the phase subtraction, preceded by an estimation of the aberration profile using areas of a hologram that have not been affected by the object. The phase subtraction methodology, developed on the ground of the thin element approximation, is very effective in the visual enhancement of phase images; however, its application to quantitative measurement of micro-objects is questionable. Therefore, in this paper we verify the possible use of the phase subtraction methodology in DHM by performing a numerical experiment, supported with the finite difference time domain method (FDTD), which allows us to identify the residual error of the correction. The FDTD computation reveals that the phase subtraction methodology is insufficient to properly remove the influence of a capillary, in particular to compensate for two effects associated with the focusing properties of the aberration: a transversal shift of the image and the change of its magnification. Nevertheless, the possibility of the visual improvement of holographic images of a living human leukemia cell using the outlined method is demonstrated.
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